DE3044970C2 - - Google Patents

Description

The invention relates to the new compound 3 ', 4'-dideoxyparomomycin, Process for its preparation and antibacterial agents against gram-positive, gram-negative bacteria and against protozoa.

The production process comprises the sulfonylation in the 4'-position of the known 6.3 ', 2'',5'',3''', 4 '''-hexa-O-acetyl-6'- O-benzoyl-penta- N-benzyloxycarbonylparomomycins, whereby the 4'-O-sulfonyl derivative is obtained. Treatment with a strong base gives 3 ', 4'- β- epoxy-penta-N-benzyloxycarbonylparomomycin. After the free hydroxyl groups have been reacted, the epoxy derivative is treated with sodium acetate, acetic acid and sodium acetate, giving 6.6 ', 6'',5'',3''', 4 '''-hexa-O-benzoyl-4'-deoxy-4'-iodo-penta-N-benzyloxycarbonylparomomycin- is obtained. From this compound by reaction with methanesulfonyl chloride in pyridine, the corresponding 6,6 ', 2'',5'',3''', 4 '''-hexa-O-benzoyl-3',4'-dideoxy-3'-Eno-penta-N-benzoyloxycarbonylparomomycin obtained. De-O-acylation of this intermediate gives 3 ′, 4′-dideoxy-3′- eno-penta-N-benzyloxycarbonylparomomycin, from which after hydrogenation of the 3 ′, 4′-carbon-carbon double bond and simultaneous removal of the N- Protective groups the desired 3 ', 4'-dideoxyparomomycin obtained and optionally isolated in the sulfate form.

Methods for producing dideoxy sugar derivatives are known; see. Bull. Chem. Soc. Japan (1979), 52, (4), 1131-1134, and U.S. Patent 40 78 138.

The process according to the invention proceeds via intermediates of the general formula:  

in the

• (1) R₁ for an acyloxy group having 2 to 5 carbon atoms or a benzoyloxy group, R₂ is an alkylsulfonyloxy, arylsulfonyloxy or aralkylsulfonyloxy group stands, R₃ for a hydrogen atom stands for X, an acyl group with 2 to 5 Carbon atoms or a benzoyl group, Y stands for a benzoyl group and Z for a benzyloxycarbonyl group stands, or
• (2) R₁ and R₃ together represent an epoxy group, R₂ represents a hydrogen atom, X and Y are the same and are hydrogen atoms, acyl groups with 2 to 5 Represent carbon atoms or benzoyl groups and Z represents a benzyloxycarbonyl group, or  
• (3) R₁ represents a hydroxy group, R₂ represents an iodine atom stands, R₃ represents a hydrogen atom, X and Y are the same and acyl groups with 2 to 5 carbon atoms or mean benzoyl groups and Z for one Benzyloxycarbonylgruppe stands or
• (4) R₁, R₂ and R₃ and the carbon atoms to which they are attached are a vinylene group, X and Y are the same and have hydrogen atoms, acyl groups 2 to 5 carbon atoms or benzoyl groups and Z represents a benzyloxycarbonyl group.
• (5) In the compound of the invention all groups R₁, R₂, R₃, X, Y and Z are hydrogen atoms.

The compound of the invention can from 6'-O-benzoyl 6.3 ′, 2 ′ ′, 5 ′ ′, 3 ′ ′ ′, 4 ′ ′ ′ - hexa-O- (C₂-C₅-acyl or -benzoyl) -penta- N-benzyloxycarbonylparomomycinen be prepared. These Starting materials can in turn be derived from paromomycin, a natural aminoglycoside antibiotic (US Pat. No. 2,916,485 and 30 65 147) by the process according to EP-PS 00 21 150 or from the same precursor using benzoylimidazole in chloroform with a shorter reaction time and with comparable yield can be produced. The production, which also falls within the scope of this invention explained in the following reaction scheme. In it A means an acyl or benzoyl group, Bz a benzyl group, Ph a Phenyl group and R is an alkyl, aryl or aralkyl radical.  

The preparation comprises the sulfonylation of the only free hydroxyl group (in the 4'-position) of the starting material ( 1 ), whereby the 4'-O-sulfonyl derivative ( 2 ) is obtained. The reaction is carried out using an alkylsulfonyl halide, an aralkylsulfonyl halide or an arylsulfonyl halide, preferably methylsulfonyl chloride, p-bromobenzenesulfonyl chloride, p-toluenesulfonyl chloride, benzylsulfonyl chloride or trifluoromethylsulfonyl chloride, in the presence of a base, preferably pyridine. The reaction of compound ( 2 ) with a strong base, suitably sodium methoxide, in methanol or chloroform provides the key intermediate ( 3 ) in which the hydroxy groups are not protected and positions 3 'and 4' are involved in an oxirane ring. This gives the possibility of selective modifications in such positions. In order to convert the epoxy group into a methylene group, the free hydroxy groups of the compound ( 3 ) are again protected by an esterification reaction, whereby the epoxy derivative ( 4 ) is obtained, which is treated with sodium iodide, acetic acid and its sodium salt. This treatment opens the oxirane ring, thereby maintaining the iodohydrin grouping in the compound ( 5 ). The 3 ', 4'-unsaturated derivative ( 6 ) is prepared from the compound ( 5 ) by reaction with methanesulfonyl chloride in pyridine and subsequent heating. De-O-acylation of compound ( 6 ) gives 3 ′, 4′-dideoxy-3′-enopenta-N-benzyloxycarbonylparomomycin ( 7 ). The hydrogenation of the carbon-carbon double bond and the removal of the N-protecting groups are achieved by catalytic hydrogenation, suitably by catalytic transfer hydrogenolysis in the presence of 10% palladium on charcoal, whereby 3 ′, 4′-dideoxyparomomycin ( 8 ) is obtained. This product can be purified by ion exchange column chromatography and converted to the sulfate form.

The mass spectrometric values (field desorption) indicate by fragmentation that both deoxygenation sites are in ring A.

3 ′, 4′-dideoxyparomomycin is an antibacterial agent against gram-positive and gram-negative bacteria and against protozoa effective. The straightest way to improve Spectrum of antibacterial activity of natural aminoglycoside Antibiotics have been the enzymatic sites Remove inactivation or sterically obstruct. It is believed that the hydroxy group is in the 3'-position in the aminoglycoside antibiotics versus an enzymatic Inactivation by resistant bacterial strains formed phosphotransferase enzymes are sensitive and that the 4'-hydroxy group also an enzymatic inactivation experienced by O-nucleotidylation (see Kirk-Othmer "Encyclopedia of Chemical Technology ", Volume 2, 3rd edition, 1978, by John Wiley and Sons, Inc.). It is expected that the Removal of these hydroxy groups a broader spectrum of activity results, although not bound by these theoretical Considerations.

The invention therefore a drug for Provided the 3 ', 4'-dideoxyparomomycin or a pharmaceutically acceptable salt thereof in admixture with one pharmaceutically acceptable diluent or carrier contains.

Biological activity

In Table I below, the antibacterial in vitro Activity tested according to the serial dilution method in the solid Medium specified. The activity of 3 ′, 4′-dideoxyparomomycin is lower than that of paromomycin, however  closes its range E. coli K12-R148, APH (3 ′) II producers and S. epidermidis PS109, AAD (4 ′) - producers, both of which are highly paromomycin resistant are a.

The therapeutic activity was subcutaneous to paromomycin tested in mice experimentally with S. aureus Smith and Shigella flexneri had been infected. 3 ′, 4′- Dideoxyparomomycin shows a slightly lower (Table II) Paromomycin activity against Staphylococcus infection. On the other hand, with a Shigella infection the activity almost three times higher.

The compound 3 ', 4'-dideoxyparomomycin is with regard to their strong resistance to the two aminoglycoside inactivators Enzymes widely in the bacterial population are widespread, and because of its excellent bioavailability very interesting.

Table I

In vitro activity of 3 ′, 4′-dideoxyparomomycin against paromomycin

Table II

Therapeutic activity in experimentally infected mice

The invention is illustrated in the following example. All temperature information are expressed in ° C.

example A. Hexa-O-acetyl-6'-O-benzoyl-4'-O-p-bromobenzenesulfonyl-penta- N-benzyloxycarbonylparomomycin

2 g 6.3 ′, 2 ′ ′, 5 ′ ′, 3 ′ ′ ′, 4 ′ ′ ′ - hexa-O-acetyl-6′-O-benzoyl-penta-N- benzyloxycarbonylparomomycin, produced according to the EP-PS 00 21 150, were dissolved in 20 ml of methylene dichloride, and the solution was treated with 4 ml of triethylamine and 0.1 g of 4-dimethylaminopyridine transferred. 1 g of p-bromobenzenesulfonyl chloride in 3 ml of methylene dichloride was added to the ice cold solution. After 2 h at 0 ° C the reaction mixture was 4 days long stirred at ambient temperature. The mixture was in Evaporated in vacuo and the residue was dissolved in chloroform, with 0.5N hydrochloric acid, water, sodium bicarbonate solution and washed water in that order. The organic  Solution was dried and evaporated, leaving 2.5 g of residue were obtained. Purification by preparative TLC analysis on silica gel plates supplied 1.5 g of the heading compound mentioned (yield 65%) in pure form. The compound has an Rf of 0.35 in TLC analysis in the solvent system toluene / ethyl acetate (volume ratio 1: 1) (Rf value of the starting product: 0.20).

B. 3 ', 4'- β- epoxy-penta-N-benzyloxycarbonylparomomycin

11 ml of 0.5N methanolic sodium methoxide solution were added to a solution of 1.1 g of the compound prepared according to A in 11 ml of methanol. The mixture was stirred at room temperature for 1.5 hours and water was added. After extraction with ethyl acetate, the aqueous phase was evaporated to dryness. The residue was redissolved in water and extracted with ethyl acetate. The organic extracts were washed with water and the solvent was evaporated. The residue was precipitated from acetone / diethyl ether, whereby 0.72 g of crude product was obtained. Purification by silica gel column chromatography eluting with chloroform / ethyl acetate / methanol (volume ratio 40: 25: 9) gave 0.45 g of the title compound (yield 60%) in pure form with an Rf value of 0.26 in the TLC analysis in the solvent system chloroform / ethyl acetate / methanol (volume ratio 40: 24: 9). Mp 126 to 128 °, [ α ] + 30.6 (c 1.044 CHCl₃), NMR spectrum (60 MHz, CD₃COCD₃) w : 3.15 (2H, s, oxirane ring protons).

Elemental analysis:

Calculated for C₆₃H₇₃N₅O₂₃: C 59.66, H 5.80, N 5.52%; Found: C 59.02, H 5.75, N 5.44%.  

C. Hexa-O-acetyl-6'-O-benzoyl-4'-O-methanesulfonyl-penta-N-benzyl- oxycarbonylparomomycin

To a solution of 13 g 6.3 ', 2'',5'',3''', 4 '''-hexane-O-acetyl-6'-O-benzoyl-penta-N-benzyloxycarbonylparomomycin in 260 ml anhydrous pyridine was added to 4.6 ml of methanesulfonyl chloride. The reaction mixture was stirred at 40 to 50 ° C for 2 hours. 30 ml of water was added at ambient temperature and the solution was evaporated. Water was added to the residue and, after extraction with chloroform, the organic extracts were washed with water, dried and evaporated in vacuo. The residue was reprecipitated from chloroform / diethyl ether / n-hexane, whereby 13.5 g of the title compound (yield 99%) were obtained in a sufficiently pure form for the further conversions. Mp 125 to 130 °, [ α ] + 27.8 (c 1.024 CHCl₃), NMR spectrum (60 MHz, CDCl₃) δ : 2.8 (3 H, s, CH₃SO₃-).

Elemental analysis:

Calculated for C₈₃H₉₃N₅O₃₃S: C 57.93, H 5.44, N 4.07, S 1.8%; Found: C 57.47, H 5.40, N 4.00, S 1.5%.

D. 3 ', 4'- β- epoxy-penta-N-benzyloxycarbonylparomomycin

13 g of the compound prepared according to C were dissolved in 130 ml of chloroform. 25 ml of a 1.45N methanolic sodium methoxide solution were added dropwise to the ice-cold solution. The solution was stirred at 0 ° for 1.5 hours and then saline was added. The mixture was extracted with ethyl acetate. The organic extracts were washed with salt water and water, dried and evaporated. The residue was reprecipitated as a white amorphous powder (9 g) from acetone / diethyl ether. Purification by preparative HPLC analysis on a silica gel column eluting with chloroform / ethyl acetate / methanol (volume ratio 40: 25: 9) gave 5 g of pure 3 ′, 4′- β- epoxy-penta- N-benzyloxycarbonylparomomycin (yield 52%) .

E. 3 ', 4'- β- epoxy-hexa-O-benzoyl-penta-N-benzyloxycarbonylparomomycin

To an ice-cold solution of 770 mg according to B and D prepared compound in 10 ml of anhydrous pyridine benzoyl chloride (1.5 ml) was added. After 1 h at 0 ° the mixture was stirred at room temperature for 24 h. 3 ml of water were added added and the solvent was reduced Pressure removed. The residue was dissolved in chloroform washed with water, dried and evaporated. The re-precipitation from chloroform / diethyl ether / n-hexane gave 765 mg the compound mentioned in the heading (yield 66%) in pure form with an Rf value of 0.43 in the TLC analysis (Silica gel) in the solvent system toluene / ethyl acetate (volume ratio 60: 40).

F. 6,6 ′, 2 ′ ′, 5 ′ ′, 3 ′ ′ ′, 4 ′ ′ ′ - hexa-O-benzoyl-4′-deoxy-4′-iodo-penta-N- benzyloxycarbonylparomomycin

760 mg of the compound prepared according to E were in 14 ml of acetone dissolved. 300 mg of sodium iodide,  25 mg of sodium acetate and 0.4 ml of acetic acid. The mixture was refluxed for 7 hours, then the solvent removed under reduced pressure. The residue was dissolved in chloroform, washed with water and dried. Evaporation of the solvent left 705 mg the compound mentioned in the heading (yield 86%) back in sufficient pure form for the subsequent implementation.

G. 6,6 ′, 2 ′ ′, 5 ′ ′, 3 ′ ′ ′, 4 ′ ′ ′ - hexa-O-benzoyl-3 ′, 4′-dideoxy-3′-eno-penta- N-benzyloxycarbonylparomomycin

Methanesulfonyl chloride (0.25 ml) was added to an ice cold solution of 700 mg of the compound prepared according to F in 7.5 ml of dry pyridine. The reaction mixture was refluxed for 45 minutes and then concentrated in vacuo. After dilution with water, it was extracted with chloroform. The extracts were washed with sodium thiosulfate solution and with water, dried and evaporated. The residue was purified by preparative TLC analysis (silica gel), whereby 375 mg of the title compound (yield 58%) were obtained in pure form. The compound had an Rf value of 0.34 in the TLC analysis (silica gel) in the solvent system toluene / ethyl acetate (volume ratio 70:30). The stain turned yellow immediately after spraying with a cold 1% potassium permanganate solution. NMR spectrum (60 MHz, CDCl₃) δ : 5.8 (2 H, m, olefinic protons).

H. 3 ', 4'-dideoxy-3'-eno-penta-N-benzyloxycarbonylparomomycin

370 mg of the compound prepared according to G were in 14 ml of 0.05N methanolic sodium methoxide solution, and the mixture was stirred at ambient temperature for 10 hours. The reaction was started by adding solid carbon dioxide and water suddenly stopped, and the solution was evaporated to dryness. Water was added to the residue and the mixture was extracted with ethyl acetate. The extracts were washed with water and evaporated, whereby 245 mg of crude product were obtained. Cleaning by preparative TLC analysis (silica gel) using chloroform / Ethyl acetate / methanol (volume ratio 40: 25: 9) as an eluent gave 135 mg of the above Compound (yield 55%) in pure form. This product had an Rf value of 0.24 in the TLC analysis (silica gel) in the same solvent system.

J. 3 ′, 4′-dideoxyparomomycin

1.4 ml of cyclohexene and 230 mg of 10% palladium on charcoal were added to a solution of 130 mg of the compound prepared according to H in 14 ml of 80% ethanol. The mixture was refluxed for 1 h, filtered and evaporated in vacuo to give 55 mg of residue. The crude product was purified on a column of Amberlite CG 50® (NH₄⁺ form, 150 to 75 µm), washed with water and eluted with increasing concentrations of ammonia (0.05 to 0.2N), whereby 30 mg of the in the title compound (yield 49%) were obtained. The product (free base) was converted to the sulfate form by adding 0.2N sulfuric acid with a pH of 6 and reprecipitating from acetone. The product was homogeneous in the TLC analysis (silica gel) in the solvent system chloroform / methanol / concentrated ammonia (volume ratio 1: 3: 2) and had an Rf value of 0.35. The mass spectrum (field desorption) (free base) showed a peak at m / e 584 (MH⁺) and fragments at 424 and 455, indicating that both deoxygenation sites are in ring A.

Claims (3)

1. 3 ′, 4′-dideoxyparomomycin of the formula: 2. A process for the preparation of 3 ', 4'-dideoxyparomomycin according to claim 1, characterized in that in known manner in each case the known 6.3', 2 '', 5 '', 3 ''',4''' - Hexa-O-acetyl-6'-O-benzoyl-penta-N-benzyloxycarbonylparomomycin of the formula: in which A represents an acyl group (acetyl), Bz represents a benzyl group and Ph represents a phenyl group, in the presence of a base with an alkylsulfonyl halide, an aralkylsulfonyl halide or a halogen-substituted arylsulfonyl halide, thereby reacting the 4′-O-sulfonyl derivative of the formula: in which A, Bz, Ph have the meanings given above and R represents an alkyl, halogen-substituted aryl or aralkyl group, is obtained that the compound obtained is then treated in methanolic or in chloroform solution with a strong base, whereby 3 ', 4′- β- epoxy-penta-N-benzyloxycarbonylparomomycin of the formula: is obtained by acylating all the hydroxyl groups with benzoyl chloride to give the 3 ′, 4′- β- epoxy-hexa-O-benzoyl-penta-N-benzyloxycarbonyl-paromomycin of the formula: in which A stands for acyl (benzoyl), is obtained by treating this compound with sodium iodide, acetic acid and sodium acetate in order to open the corresponding 6,6 ', 2'',5'',3''', 4 by opening the oxirane ring ′ ′ ′ -Hexa-O-benzoyl-4′-deoxy-4′-iodo-penta-N-benzyloxycarbonylparomomycin of the formula: where A is acyl (benzoyl), to obtain that the corresponding 3 ', 4'- unsaturated derivative, ie the compound 6,6', 2 '', 5 'from this compound by treatment with methanesulfonyl chloride in pyridine at reflux temperature ′, 3 ′ ′ ′, 4 ′ ′ ′ - hexa-O-benzoyl-3 ′, 4′-dideoxy-3′-eno-penta-N-benzyloxycarbonylparomom-ycin of the formula: in which A and Bz have the meanings given above, that 3 ', 4'-dideoxy-3'-eno-penta-N-benzyloxycarbonylparomomycin is obtained from this compound after de-O-acylation by treatment at room temperature with dilute methanolic sodium methoxide solution of the formula: is obtained from which the desired 3 ', 4'Dideoxyparomomycin of formula I is obtained by catalytic transfer hydrogenolysis in the presence of 10% palladium on charcoal and cyclohexene in 80% ethanolic solution and by treatment at reflux temperature for one hour, and that the product is obtained by ion exchange Column chromatography on Amberlite CG 50® (NH₄⁺ form; 150 to 75 µm) using increasing concentrations of ammonia (0.05 to 0.2N) as the eluent and optionally isolated in its sulfate form. 3. Medicament containing 3 ', 4'-dideoxyparomomycin or a pharmaceutically acceptable salt thereof mixed with a pharmaceutically acceptable diluent or carrier.